2.1. Purification of Biologically Active Proteins and Polypeptides
In recent years, a wide variety of important biologically active molecules has been isolated from natural sources or produced by recombinant DNA technology. Many of these molecules are polypeptides or proteins which exert their biological effects by binding to specific cellular receptors.
To purify these polypeptides and proteins, conventional methods such as preparative electrophoresis, gel filtration chromatography, salt or organic solvent fractionation, ion exchange chromatography and the like have often been employed. The problem with the use of such conventional methods is that they are not generally specific for the protein of interest. As a consequence, many of these methods must generally be used in combination before a substantially pure protein can be obtained. Because there are losses due to analytical sampling and incomplete recovery at each purification step, the overall yields are often poor in such multistep purification procedures.
More recently, improved results have been obtained by using immunoaffinity columns containing polyclonal or monoclonal antibodies specific for a protein that is to be purified. For example, Kung, U.S. Pat. No. 4,476,049, has used immobilized monoclonal antibodies specific for the carboxyl terminus of recombinant human immune interferon to purify that protein. The use of immunoaffinity columns can lead to highly purified or even homogeneous protein preparations, but proteins purified in this way may not have their full intrinsic bioactivity.
Pestka, U.S. Pat. No. 4,623,621, has disclosed that many important biologically active polypeptides and proteins can aggregate, forming dimers, trimers and higher oligomeric forms. When such aggregation occurs, the polypeptide or protein may have either lower biological activity or no activity at all. Furthermore, such aggregated material may have the potential for causing deleterious side effects such as antibody production in patients, if it is used therapeutically. Immunoaffinity columns do not necessarily discriminate between the aggregated and unaggregated (monomeric) forms of polypeptides and proteins.
The probability that aggregation of desired polypeptides and proteins will occur is substantially increased when they are produced by recombinant DNA techniques in host organisms and isolated from cell lysates, under conditions and in concentrations that do not occur when they are produced under natural conditions. These conditions can favor aggregation through intermolecular disulfide bridges, other covalent bonds or non-covalent interactions.
2.2. Cloning and Expression of IL-2 receptors
Because most if not all biologically active polypeptides and proteins act b binding to specific cellular receptors, there have been numerous efforts in recent years to characterize these receptors. Recently, the methods of recombinant DNA technology have been brought to bear on the problem of producing receptor molecules for study. Significant progress in this area has been made in the cloning and expression of genes coding for receptors for interleukin-2.
Interleukin-2 (IL-2) is a hormone-like protein growth factor secreted by certain T lymphocytes after antigenic or mitogenic stimulation. The actions of IL-2 are mediated through the binding of the IL-2 protein to specific high affinity receptors which are present in the membranes of activated, but not resting, lymphocytes.
Miller et al. [J. Immunol. 134:4212 (1985)] and Shimuzu et al. [Nucleic Acids Res. 13:1505 (1985)] have cloned and expressed cDNA prepared from murine mRNA coding for the mouse IL-2 receptor. Leonard et al. [Nature 311:625 (1984)] and Nikaido et al. [Nature 311:631 (1984)] have described similar studies based on the human IL-2 receptor. In all of these studies, plasmids bearing IL-2 receptor-encoding inserts were used to transfect COS cells. The transfected cells were then found by specific antibody or IL-2 binding to have the expressed IL-2 receptors in their cell membranes. There was no indication in these studies that receptor molecules had been secreted into the culture medium.
More recently, Treiger et al. [J. Immunol. 136:4099 (1986)] have disclosed the expression of a secreted form of the human IL-2 receptor. This secreted form of the receptor was produced by cleaving a plasmid containing a full length IL-2 receptor cDNA with the Nae I restriction endonuclease, to delete nucleic acid sequences coding for the intracytoplasmic segment and most of the putative transmembrane segment of the complete receptor molecule. The resulting modified cDNA was then transfected into and expressed in modified mouse L cells. The polypeptide thereby produced was secreted into the medium, because its "anchor" components had been deleted.